Ground truth annotation workflow for mitochondria. (A) Example to illustrate the sequential steps used with Ilastik Carving module to generate the ground truth annotation for a mitochondrion in Cell 1 HEK293A prepared by chemical fixation and visualized with ∼5 nm isotropic resolution. Coarse annotations for background (yellow) and object (blue) drawn in broadly spaced consecutive planes of the stack were used to seed the Ilastik Carving module from which a binary mask spaced along adjacent planes spaced 5 nm in the z-stack and corresponding to the mitochondria ground annotation was generated (magenta). Manual corrections using VAST are used as needed, to remove incorrectly assigned pixels, in this example corresponding to an adjacent ER (white arrow). Scale bar, 500 nm. (B) Volume rendering corresponding to the ground truth annotation of the mitochondrion shown in A.